Four to ten carbon alkanes have many uses in our society, particularly as fuels and as feedstock for more complex chemicals. Most, however, are produced from petroleum, a dwindling reserve whose use creates significant ecological impact.
Butane (C4), for example, is mainly used for gasoline blending, as a fuel gas, either alone or in a mixture with propane, and as a feedstock for the manufacture of ethylene and butadiene, a key ingredient of synthetic rubber. Isobutane is primarily used by refineries to enhance the octane content of motor gasoline.
Very pure forms of butane, especially isobutane, can be used as refrigerants and have largely replaced the ozone layer-depleting halomethanes, for instance in household refrigerators and freezers. The system operating pressure for butane is lower than for the halomethanes, such as R-12, so R-12 systems such as in automotive air conditioning systems, when converted to butane will not function optimally.
Butane is also used as lighter fuel for a common lighter or butane torch and is sold bottled as a fuel for cooking and camping, and cordless hair irons are usually powered by butane cartridges.
In industry, hexanes (C6) are used in the formulation of glues for shoes, leather products, and roofing. They are also used to extract cooking oils from seeds, for cleansing and degreasing a variety of items, and in textile manufacturing. A typical laboratory use of hexanes is to extract oil and grease contaminants from water and soil for analysis. Since hexane cannot be easily deprotonated, it is used in the laboratory for reactions that involve very strong bases, such as the preparation of organolithiums, e.g. butyllithiums are typically supplied as a hexane solution. In many applications (especially pharmaceutical), the use of n-hexane is being phased out due to its long term toxicity, and often replaced by n-heptane, which will not form the toxic metabolite hexane-2,5-dione.
Octanes (C8) became well known in American popular culture in the mid- and late-sixties, when gasoline companies boasted of “high octane” levels in their gasoline advertisements. Thus, it too is useful in fuels. Decane (C10) undergoes combustion reactions in a similar fashion to other alkanes.
Thus, we can see that there are many important uses for low carbon number alkanes and the demand for C4+ alkanes is not expected to diminish any time soon. Yet as products of petroleum refining, the production of such alkanes contributes significantly to environmental degradation, and as our hydrocarbon resources continue to dwindle in availability, the alkanes can only be expected to increase in price over the long term.
There is also need for alcohols and acids of the C-4+ class such as butyrate, hexanoic acid, etc. and the corresponding alcohols that are used in many chemical processes. Chemical processes are known for interconversions among the C-4+ series of carbon compounds and used by the petrochemical industry, so a source of a particular reduced C-4+ compound can be useful for a variety of potential industrial processes.
Thus, what are needed in the art are biological sources for these important alkanes, and microbial production is being investigated in that regard. Unfortunately, not many bacteria make butane or hexane, at least not in significant amounts, and some of the bacteria that do are obligate anaerobes, which are difficult and expensive to culture.
Professor David Mullin, and his team have discovered a new bacteria, called Tu-103, a butane-producing bacteria that lives on glycerol—a byproduct of biodiesel synthesis, or on cellulose—a waste product in abundant supply from e.g., old newspapers. The microbe is unique because it can do this in the presence of oxygen, unlike some other types of bacterium, which means less expensive production techniques would be required than for most obligate anaerobes. However, little is known about this bacteria because details are being kept as a trade secret, and future patents may also prevent its use.
Nonetheless, the existence of such organisms has generated renewed interest in solventogenic bacteria, such as Clostridia, because it is anticipated that additional strains will be discovered that have some degree of tolerance to oxygen, removing some of the difficulties in using these organisms for the bioproduction of desired chemicals. Alternatively, increasing exposure to oxygen may induce some degree of oxygen tolerance, and/or random mutagenesis could result in such changes.
Clostridium acetobutylicum, for example, is an anaerobic, spore-forming prokaryote that produces the solvents butanol, acetone, and ethanol. The desired product of the C. acetobutylicum fermentation is butanol, which has superior fuel characteristics to ethanol, such as higher energy content and lower water miscibility. The C. acetobutylicum genome has been completely sequenced and annotated, and methods for genetic deletions and gene overexpression have been developed, making it even more attractive organism for further strain development. Clostridia can also grow on a variety of substrates, from simple pentoses and hexoses to complex polysaccharides.
The metabolism of C. acetobutylicum is typically biphasic in batch culture—the cells first produce acetate and butyrate, and later—butanol, acetone, and ethanol. During growth, the production of acids lowers the pH of the culture, which combined with butyrate accumulation causes a shift in metabolism towards solvent production.
As solvents are produced, the acids are typically re-assimilated and converted into solvents. With initiation of solvent formation, the cells commit to their sporulation program. In continuous culture or upon consecutive vegetative transfers, cells may degenerate whereby they become asporogenic and lose the capability to produce solvents. In this organism, the degeneration process is due to the loss of the pSOL1 megaplasmid, which carries the key solvent formation genes in the so-called sol locus made up of the sol operon (aad-ctfA-ctfB) (coding for the enzymes AAD and CoAT) and the adc gene (coding for the enzyme AADC, FIG. 5). The use of pSOL1 mutants can be beneficial in certain instances, reducing competition for carbon resources, and driving metabolism towards desired products.
What are needed in the art are additional methods of making C4-10 compounds using microbes. A method using some of the advantages of solventogenic bacteria, such as Clostridia may be of benefit as well.